Incubation media modify silver nanoparticle toxicity for whitefish (Coregonus lavaretus) and roach (Rutilus rutilus) embryos

Figures & data

Figure 1. TEM images of NM-300 K (AgNPs) in the stock dispersion. The nanoparticles had an average size of 14.4 ± 2.5 nm

Figure 2. Dose-response curves and data points of AgNP exposure to C. lavaretus in ASTM (blue), ISO (red), and Lake Mondsee water (green) and R. rutilus in Lake Mondsee water (black) for (a) lethal and (b) non-lethal adverse effects. Non-lethal adverse effects include adverse factors for the embryo such as edema or phenotypic defects of the head, tail, or yolk. Coloration around each line display the respective 95% CIs. Open symbols depict the observed effects per replicate (C. lavaretus: ASTM □, ISO ○, Lake Mondsee water Δ; R. rutilus: Lake Mondsee water ◊). Black horizontal dashed line displays the median lethal/non-lethal adverse effect line. n = 3, expect for ASTM 0.1 mg/L and 0.5 mg/L, ISO 2 mg/L, and Lake Mondsee water 0.5 mg/L with n = 2, because mortality in the internal plate control exceeded 25% in one replicate

Figure 3. Average mortality over time for C. lavaretus and R. rutilus embryos after exposure (8 weeks and 9 days, respectively) to AgNPs ranging from Control (0) to 5 mg/L (nominal concentration) and stabilization agent NM-300 K DIS in ASTM, ISO, and Lake Mondsee water medium. n = 60 for each data point, except for ASTM 0.1 mg/L and 0.5 mg/L, ISO 2 mg/L, and Lake Mondsee water 0.5 mg/L with n = 40, because the mortality in the internal plate control exceeded 25% in one well plate

Figure 4. Developmental time (± SD) until reaching the eye-point stage for C. lavaretus embryos. Number of embryos surviving until this developmental stage in the respective AgNP exposure sample are displayed in each bar. Asterisks* indicate significant difference to the control at p < .05 Bonferroni adjusted Mann-Whitney U-test). Missing data points (bars) indicate that the mortality was at 100% before the eye-point stage was reached. n = 60 for each data point, except for ASTM 0.1 mg/L and 0.5 mg/L, ISO 2 mg/L, and Lake Mondsee water 0.5 mg/L with n = 40, because mortality in the internal plate control exceeded 25% in one well plate

Table 1. Mode of the particle size distribution function in nm (± SD) of primary AgNPs (NM-300 K) in ASTM, ISO, Lake Mondsee water, and MilliQ after 0, 24, 96, and 168 hr determined by TEM. Different letters indicate significant difference between test media for each time point (Bonferroni adjusted Mann-Whitney U-test). n.a. = not analyzed

Time (hr)	ASTM (nm)	ISO (nm)	Lake Mondsee water (nm)	MilliQ (nm)
0	13.9 ± 3^a	14.5 ± 3.3^b	14.3 ± 3.3^ab	14.4 ± 2.5^ab
24	14.3 ± 2.8^a	13 ± 4.1^a	14.3 ± 2.5^a	n.a.
96	12.3 ± 2.7^a	11.2 ± 2.9^b	11.4 ± 2.7^ab	n.a.
168	15.3 ± 2.1^a	13.7 ± 3.6^a	9.8 ± 3^b	15.3 ± 2.1^a

Figure 5. (a) Total silver as a measure for AgNP (n = 1 with 10 replicated measurements; nominal concentration: 100 µg/L) and (b) concentration of Ag⁺ dissolved from AgNPs assessed with filters of 50 kDa (n = 2 with 10 replicated measurements each; nominal concentration: 100 µg/L) in ASTM, ISO, and Lake Mondsee water after 0, 24, 96, and 168 hr. Dissolved silver from AgNP in MillQ water was only measured at 0 hr. Samples were acidified with HNO₃ to pH 1–2, frozen and stored at −20°C until analysis with ICP-MS. Error bars represent standard deviation

Table 2. Silver species distribution (in %) according to thermodynamic calculations (based upon Gustafsson 2017) for low (0.5 µg/L) and high (5 µg/L) concentrations of Ag⁺ released from AgNPs in ASTM, ISO, and Lake Mondsee water

	ASTM		ISO		Lake Mondsee water
	low	high	low	high	low	high
Ag(OH)2^−	< 0.1%	< 0.1%	< 0.1%	< 0.1%	< 0.1%	< 0.1%
Ag^+	71.6%	71.6%	9.5%	9.7%	64.8%	64.8%
AgCl(aq)	25%	25%	66.7%	66.6%	34.3%	34.3%
AgCl2^−	0.5%	0.5%	23.6%	23.6%	0.9%	0.9%
AgCl3^2-	< 0.1%	< 0.1%	0.1%	0.1%	< 0.1%	< 0.1%
AgOH(aq)	< 0.1%	< 0.1%	< 0.1%	< 0.1%	< 0.1%	< 0.1%
AgSO4^−	2.9%	2.9%	0.1%	0.1%	0.1%	0.1%

Gustafsson, J. P. 2017. Visual MINTEQ. Page https://vminteq.lwr.kth.se/, last visited: November 2017.

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Data Availability Statement

All supportive data and associated metadata are available in the supplementary material.